Mycotoxins are secondary metabolites produced by numerous mold species, principally belonging to the genera Penicillium, Aspergillus and Fusarium. Currently, more than 300 mycotoxins are known, including, among others, the aflatoxins, ochratoxins, trichothecenes, fumonisins, zearalenone, citrinin and patulin. Although the acute and subacute toxicity of some mycotoxins is well known, the effects of long-term ingestion is of greater concern, as the small quantities ingested with foods on a continual basis accumulate in the body and can have, in some cases, mutagenic and carcinogenic effects (Martin et al., 1990. Revista de Agroquímica y Tecnología de los Alimentos, 30:315-332).
Ochratoxins are a group of mycotoxins produced on different substrates by some species of fungi, noteworthy among them are the genera Aspergillus and Penicillium. Although there are various types of ochratoxins, ochratoxin A (OTA) is the most toxic. Because these molds are capable of growing in a wide variety of foods, OTA can be found in meat and dairy products, cocoa, fruit, cereals, coffee, olive oil, nuts, spices, baby food and fermented products such as wine and beer among others, under highly variable humidity, pH and temperature conditions (Engelhardt G. et al., 1999. Adv. Food Sci. 21, pp. 88-92; Romani S., et al., 2000. Journal of Agricultural and Food Chemistry 48, pp. 3616-3619).
OTA is a derivative of isocoumarin linked to L-β-phenylalanine through the carboxyl group. Chemically, it is N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)carbonyl]-L-phenylalanine, a chlorinated dihydrocoumarin linked via a carboxyl group by an amide bond to a L-β-phenylalanine molecule. Other types of ochratoxins are ochratoxin B (OTB), a less toxic, nonchlorinated derivative of OTA, ochratoxin C (OTC), OTA ester, with little toxic potential, ochratoxin α (OTα) and ochratoxin β (OTβ), hydrolysis products of OTA and OTB that do not have the phenylalanine molecule and are not considered toxic, respectively (Pavón et al., 2007. RCCV Vol. 1 (2)).
In recent years, ochratoxins and in particular ochratoxin A, have received particular attention, due to their high toxicological potency. Ochratoxin A, which is noticeably nephrotoxic, has been related to serious illnesses, such as “Balkan endemic nephropathy” or urinary tract tumors in humans and “spontaneous nephropathy in pigs” or “avian nephropathy” in animals. In addition, studies carried out with animals and in human cell lines have revealed its carcinogenic, genotoxic, immunotoxic, hepatotoxic, neurotoxic and teratogenic properties (Kuiper-Goodman, T. 1996. Food Addit. Contam. 13, pp. 53-57) and it has been detected in human blood after the intake of foods contaminated with it (Petkova-Bocharova, T. et al., 1988. Food Addit.Contam. 5, pp. 299-301).
Therefore, in order to ensure the health of consumers exposed to this mycotoxin, the European Union has established limits for the OTA concentration allowed in cereals, raisins, roasted coffee whether beans or ground, instant coffee, wines and grape musts, and spices. The limits vary according to the raw material, but are within a range of 2-10 μg/kg. In the case of dried vine fruits the maximum level is 10 μg/kg and the limit for unprocessed cereals is 5 μg/kg, whereas for processed cereal products used for direct human consumption it is 3 μg/kg. However, a limit of less than 0.5 μg/kg has been established for cereal-based processed foods when such foods are intended for infants and young children (European Commission Regulation (EC) No 1881/2006 of 19 Dec. 2006). The World Health Organization (WHO), on its part, has proposed 5 μg/kg as the maximum limit for OTA in cereals.
For spices and licorice, Regulation (EU) No 105/2010 establishes temporarily and for the first time a maximum content of ochratoxin A, which enters into effect this July and will be stricter as of 2012. In the case of spices the maximum concentration of 30 μg/kg is established from 1 Jul. 2010 to 30 Jun. 2012, and 15 μg/kg as of 1 Jul. 2012. The spices considered are: Capsicum spp. (fruit from such genus whether dry, whole or pulverized, including chili peppers, chili powder, cayenne and paprika), Piper spp. (fruit, including white and black pepper), Myristica fragrans (nutmeg), Zingiber officinale (ginger), Curcuma longa (turmeric), and spice blends containing one or more of the aforementioned spices.
Due, therefore, to the fact that OTA is a real problem in the food sector due to its toxicity and high presence in a large number of foods and beverages, it is necessary to reduce the levels of this mycotoxin present in food products. In this context, reliable methods capable of degrading this mycotoxin are sought.
In the coffee industry, for instance, solvent decaffeination has been reported to significantly reduce OTA levels (Heilmann W. et al., 1999. Eur. Food Res. Technol. 209, pp. 297-300); in addition, the use of ozone treatments is also proposed as a detoxification method for OTA-contaminated beans (McKenzie K. S. et al., 1997. Food Chem. Toxicol. 35, pp. 807-820). Additionally, various studies have been carried out in wine cellars to reduce the presence of OTA in wine musts and wines, among them being different decontamination procedures based on physical-chemical elimination of the toxin (Castellari M. et al., 2001. J. Agric. Food Chem. 49, pp. 3917-3921; Dumeau F., and Trioné D., 2000. Rev. Fr. Oenol. 95, pp. 37-38; García-Moruno E. et al., 2005. Am. J. Enol. Vitic. 56, pp. 73-76).
The use of physical or chemical methods for mycotoxin decontamination can eliminate, besides mycotoxin, many substances important from the organoleptic or nutritional point of view. Therefore, methods for the biological degradation of toxins are currently a very promising approach.
Regarding the biological decontamination of OTA, the scientific literature contains reports of enzymes with carboxypeptidase A (CPA) activity, such as bovine pancreatic CPA (Sigma), capable of degrading OTA. These enzymes hydrolyze the amide bond in the OTA molecule to yield L-phenylalanine and ochratoxin α (OTα) (Pitout M. J. 1969. Biochem Pharmacol 18, pp. 485-491).
It was later reported that some microorganisms have a similar mechanism of action in OTA degradation to that used by CPA enzymes, such as Phenylobacterium immobile (Wegst W. and Lingens F. 1993. FEMS Letters 17, pp. 341-344), Acinetobacter calcoaceticus, (Hwang C. A., and Draughon F. A. 1994. J. Food Prot. 57, pp. 410-414) or Aspergillus niger (Abrunhosa L. and Venancio A. 2007. Biotechnol. Lett. 29, pp. 1909-1914). It is also known that some strains of Rhodococcus are able to degrade a wide variety of organic compounds, such as Rhodococcus erythropolis which has recently been seen capable of degrading aflatoxin B1, a mycotoxin that is structurally different from OTA. (Teniola O. D. et al., 2005. Int. J. Food Microbiol. 105, pp. 111-117).
However, although the results obtained have important implications for food safety, none of the microorganisms mentioned in the preceding paragraph and able to degrade OTA are used in the food industry.
Therefore, although different treatments based on physical, chemical and biological methods to decrease ochratoxin A levels have been described, until now none of the treatments described can be used for OTA detoxification of foods.
Physical-chemical washes, treatments with absorbent materials, solvent extraction, etc., are among the physical-chemical processes commonly used. These methods are costly and may also eliminate various nutrients or compounds that are important from an organoleptic point of view. Furthermore, no biological treatments used to lower OTA content in foods, beverages and animal feeds are currently in existence, as none of the microorganisms that have been described and capable of degrading OTA are related to foods.
Consequently, there is a manifest difficulty with finding a suitable method for ochratoxin A degradation in food products such that the food properties are unaltered from both the organoleptic and nutritional point of view.